The elucidation of molecular mechanisms of aging is essential to fighting against age-related diseases and improving the quality of life of elderly people. The long-term goal of this project is to identify changes at the protein level associated with aging and longevity using animal models. A defect in klotho gene expression leads to a syndrome resembling human aging. In contrast, mice that overexpress Klotho enjoy an extended lifespan, indicating that Klotho is an aging-suppressor gene that can delay aging when overexpressed and accelerate aging when disrupted. Klotho protein functions as a hormone-like substance that inhibits insulin and IGF-1 signaling, consistent with accumulating genetic evidence that inhibition of insulin-like signaling is an evolutionarily conserved mechanism for extending lifespan from worms to mammals. Using mice with different Klotho expression levels and other established mouse models for extended longevity, proteomic changes associated with the acceleration and deceleration of aging will be identified. The specific aims are to: (i) Identify phosphorylation and modifications of signaling proteins regulated by insulin/IGF-1 signaling in mouse tissues using protein microarray technology. (ii) Identify protein-protein interactions that regulate insulin/IGF-1 signaling and aging. Preliminary data suggest that Klotho, putative Klotho receptor, IGF-1 receptor and other unidentified proteins form a complex on the cell surface, thereby regulating activity of insulin/IGF-1 signaling and eventually aging. The components of this multi-protein complex will be identified using immunoprecipitation and 2-dimensional gel fingerprint analysis followed by mass spectrometry-based protein sequencing. (iii) Develop a new diagnostic method to distinguish aging states of individual animals by profiling serum and tissue proteins using mass spectrometry. Mathematical algorithms will be developed that extract a set of biomarkers to specify different aging states. These studies are expected to provide critical information that promotes a better understanding of the molecular mechanisms of aging and longevity. [unreadable] [unreadable]